Growth of endochondral skeletal structures in skull, trunk and limbs is governed by the activity of the growth plates where chondrocytes proliferate, undergo maturation and hypertrophy and are replaced by bone. Functioning of the growth plates in turn depends on their unique extracellular matrix composed of aggrecan, collagen II and other macromolecules, and abnormalities in matrix gene expression or structure cause growth plate malfunction and chondrodysplasias, including dwarfism. There have been significant advances in growth plate and matrix biology and pathology in recent years, but much remains to be understood and discovered. The retinoic acid receptors a, [unreadable] and ? (RARa, RAR[unreadable] and RAR?) are nuclear hormone receptors that regulate numerous fundamental processes and act as ligand-less transcriptional repressors or ligand-dependent transcriptional activators. Studies conducted a decade or so ago showed that mice lacking RAR genes have skeletal defects, but detailed understanding of RAR roles has been lacking since. To study skeletal-specific RAR function, we created conditional RAR mouse mutants. We find that mice lacking RARa and RAR? (or RAR[unreadable]/RAR?) genes in cartilage exhibit severe growth retardation. Their growth plates are defective and, importantly, display a major drop in aggrecan expression and content. Mice lacking RARa and RAR[unreadable], however, are normal, suggesting that RAR? is essential. Indeed, we find that RAR? is the most strongly expressed RAR in mouse growth plates, and its expression characterizes the proliferative and pre-hypertrophic zones where aggrecan is strongly expressed also. Biochemical analysis shows that those zones are devoid of active endogenous ligands, signifying that the RARs operate as ligand-less factors. Studies with mouse chondrocyte cultures indicate that (a) RAR? over-expression enhances aggrecan expression;(b) RAR? exerts its function in cooperation with transcriptional co-repressor Zac1;and (c) RAR action on aggrecan expression is indirect and involves Sox proteins. Our central hypothesis is that RARs, and RAR? in particular, exert previously unappreciated roles in growth plate function and promote aggrecan expression and content. Ligand-less RAR repressor function would lead to stimulation of Sox expression;this, in turn, would increase aggrecan expression. Our specific goals are: (i) to determine whether RAR function in growth plates is largely, if not exclusively, due to RAR?;(ii) to define the molecular mechanisms by which RAR? regulates aggrecan gene expression via Sox proteins;and (iii) to characterize the roles of co-repressor Zac1. The results of the project will produce fundamentally new insights in skeletal and growth plate biology. The RARs represent a critical but currently understudied area of skeletal biology, and the project will begin to bridge this glaring gap. Because RAR function is susceptible to experimental and pharmacologic manipulations, the project will also lead to envision future ways in which such treatments could be exploited therapeutically to rectify chondrocyte behavior and matrix homeostasis and restore function in skeletal growth deficiencies and related pathologies. PUBLIC HEALTH RELEVANCE: The process of skeletal growth during prenatal and postnatal life is critical for establishment of a functioning skeleton, and this process is susceptible to congenital or acquired diseases including dwarfism. The mechanisms regulating skeletal growth remain ill defined and understood. This project focuses on molecular factors acting in the cell nucleus to regulate gene expression, and promises to shed new light into molecular mechanisms of growth that could be exploited therapeutically in the future to correct and restore skeletal growth and function in affected individuals.